Stacking nucleic acid and methods for use thereof

Abstract

The present invention provides a novel modified oligonucleotide monomer useful in molecular biological techniques such as capture and/or detection of nucleic acids, amplification of nucleic acids and sequencing of nucleic acids. The modified oligonucleotide monomer comprises an intercalator that can intercalate into an antiparallel duplex from the major groove.

1 . A modified oligonucleotide monomer SNA with the general structure:
   X—B-L-I
   wherein   X is a backbone monomer unit that can be incorporated into the backbone of an oligonucleotide or an oligonucleotide analogue,   B is a nucleobase, a pyrimidine or purine analog or a heterocyclic system containing one or more nitrogen atoms   L is a linker and   I is an intercalator comprising at least one essentially flat conjugated system and wherein the length of linker is between 5 and 15 angstroms.   
     
     
         2 . The monomer of  claim 1  further comprising a conjugator K between B and L or between L and I:
   X—B—K-L-I
 
   X—B-L-K—I
 
 
     
     
         3 . The X—B-L-I monomer of  claim 1  being described by
   X—B—CH 2 O(CH 2 ) n —I
 
 wherein n is 5 or 6. 
 
     
     
         4 . The X—B—K-L-I monomer of  claim 2  being described by
   X—B—K—(CH 2 ) n NHCO(CH 2 ) m CO—I,
 
 wherein n is between 1 and 3 and m is between 1 and 3 
 
     
     
         5 . The X—B-L-K—I monomer of  claim 2  being described by
   X—B—(CH 2 ) m —O—(CH2-) n —K—I
 
 wherein m is 1 and n is 3 or 4 
 
     
     
         6 . The monomer of  claims 2 ,  4  and  5 , wherein K is ethynyl 
     
     
         7 . The SNA monomer of any of the preceding claims, wherein X—B is either a DNA or RNA unit. 
     
     
         8 . The SNA monomer of any of  claims 1 - 7  adapted for enzymatic incorporation into an oligonucleotide. 
     
     
         9 . The SNA monomer of any of  claims 1 - 7  adapted for incorporation into an oligonucleotide using standard oligonucleotide synthesis 
     
     
         10 . An oligonucleotide comprising the SNA monomer of any of  claims 1 - 7 . 
     
     
         11 . Use of the SNA monomer adapted for enzymatic incorporation of  claim 8  as substrate for a polymerase. 
     
     
         12 . Use of the oligonucleotide comprising the SNA monomer of  claim 10  as primer or template in a polymerase chain reaction (PCR). 
     
     
         13 . A method comprising the steps of
 a. Providing a template nucleic acid   b. Providing a first primer oligonucleotide   c. Providing a polymerase   d. Providing a nucleotide triphosphate mixture   e. Mixing the components of steps a-d and providing conditions that allow the primer to anneal to the template.   f. Under conditions allowing primer extension, extending the first primer oligonucleotide annealed to the template   wherein the first primer oligonucleotide comprise a SNA monomer and/or   wherein the template nucleic acid comprise a SNA monomer and/or   wherein the nucleotide triphosphate mixture comprise a SNA monomer adapted for enzymatic incorporation into an oligonucleotide   
     
     
         14 . The method of  claim 13  further comprising the steps of
 g. Providing a second primer oligonucleotide, which is complementary to the first extension product of step f 
 h. Denaturing the product of the step f 
 i. Under conditions allowing primer extension, extending the second primer oligonucleotide annealed to the first extension product